title: Caprine blastocyst formation following intracytoplasmic sperm injection and defined culture.
abstract: Experiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2-6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM199 supplemented with 10 micrograms each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5 degrees C) for 4.5 h during transportation. Then, oocytes were transferred into 75 microliters of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5 degrees C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37 degrees C water bath for 15 s. Motile fractions were selected by swim-up, then incubated for 90 min in TALP with 10 micrograms heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 microliter) were added to 10 microliters medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM199 + 20% FBS at 37 degrees C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 microliters of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.
authors.affiliation: Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens 30602, USA.
authors.forename: L
authors.initials: L
authors.lastname: Keskintepe
authors.forename: P C
authors.initials: PC
authors.lastname: Morton
authors.forename: S E
authors.initials: SE
authors.lastname: Smith
authors.forename: M J
authors.initials: MJ
authors.lastname: Tucker
authors.forename: A A
authors.initials: AA
authors.lastname: Simplicio
authors.forename: B G
authors.initials: BG
authors.lastname: Brackett
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002970:Cleavage Stage, Ovum / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006041:Goats / Q000196:embryology* / Q000502:physiology; D006821:Hyaluronoglucosaminidase / Q000502:physiology; D007476:Ionophores / Q000494:pharmacology; D007986:Luteinizing Hormone / Q000494:pharmacology; D008297:Male; D009865:Oocytes / Q000502:physiology*; D009924:Organ Culture Techniques; D013094:Spermatozoa / Q000502:physiology*
chemical_list: D007476:Ionophores; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D006821:Hyaluronoglucosaminidase